Immunological changes during bed-rest: the role of countermeasures
  1. 2010 • BRAG - Bed Rest with Artificial Gravity
Life Sciences:
  • Immunology and Haematology
MEDES Space Clinic, Toulouse, France
Oliver Angerer
A. Choukèr (1), I. Kaufman (1), S. Baatout (2), G. Schelling (1), S. Kreth (1), N. Montano (3), M. Vogeser (1), M. Thiel (1)
Department of Anaesthesiology
Hospital of the Ludwig-Maximilians-University
Marchioninistrasse 15
81377 Munich
Boeretang 200
2400 Mol
University of Milan
Via Festa del Perdono, 7
20122 Milan

Short- (STBR) [mid- (MTBR) and long-term bed rest (LTBR)] will induce psycho-neuro-endocrine, metabolic and physical stress responses which will all affect the human immune system with increasing intensity. 

see Figure 1

Countermeasures, as applied by nutrition or physical means (vibration with or without exercise, artificial gravity) will modulate BR induced alterations of the specific (adaptive) and non-specific (innate) parts of the immune system.

see Figure 2

Consequences of long-term confinement and hypobaric hypoxia on immunity in the Antarctic Concordia environment (CHOICE-study)
CONCORDIA winter-over 2009
CONCORDIA winter-over 2010

CoSi-500 Study - Confinement for 500 days - Evaluation of Stress and Immunity
2009 Mars105
2010 Mars500

International Space Station 2005 - current

Study group

This is a prospective study including 8-12 male healthy volunteers who are exposed to simulated microgravity without and with defined CMs for 5, 21 and 60 days. In the short-term BR group we will assess the effects of simulation of weightlessness and those of artificial gravity.

Control groups

In dependence upon the performance of the study with a within- or a between-subject design, control groups are either the groups prior to start of the BR (for a within-subject design) or the group not subjected to a CM (for a between subject design). In the case of a between subject design, volunteers might be switched from one group to another one (cross-over design as to be determined by ESA board scientists).

Rationale and description of variables and methods

Almost all variables and methods described below are routinely applied in clinical and experimental research in our clinic and laboratory. In addition, collaboration has been initiated with other European research institutions to add more knowledge by new methods and applications (Dr. Baatout, SCK-CEN, Belgium; Prof. Montano, University of Milan, Italy). Most of these parameters and methods have been continuously improved and successfully applied in the aforementioned Earth-bound studies, in the 8th DLR Parabolic Flight Campaign and currently on the ISS, as well as in various clinical trials. Moreover, by the addition of new and innovative parameters to an existing “rack-set” repeatedly used in previous Earth-bound studies and on the ISS, we will ensure a new insight into the adoption process of humans during microgravity. Finally, an added value is to be obtained by the comparison of the newly to be determined results with those measured in our previous studies. The variables that are to be measured will also allow investigating more recently discovered stress response mechanisms (e.g. endocannabinoids) and comparison with routinely measured stress hormones (e.g. saliva cortisol) and urine catecholamine.

Immunological monitoring:

a) Cell-Counts (EDTA plasma): Concentration of lymphocytes, polymorphonuclear leukocytes (PMNL) and monocytes, Coulter multisizer, UK).
b)  ß2-integrins (heparinised blood:. The expression of adhesion molecules (ß2-integrins, CD18/CD11b) on PMNL as a marker of activation (Flow cytometry, Becton Dickinson).
c) Cytotoxic and bactericidal properties (PMNL separated from heparinised blood): Analyses of cytotoxic phagocyte functions by H2O2 production occurring spontaneously and following stimulation. As a measure of the bactericidal capacity of phagocytes, adhesion, phagocytosis and phagocytosis-associated H2O2 production will be determined.
d) Analyses of the functional role of specific adenosine receptors by assessment of the concentration-dependent effects of agonist and antagonist of A1, A2A/B and A3 receptors on the cytotoxic and bactericidal functions.
e) Lymphocyte subpopulations (heparinised blood): Percentages of functionally different populations of lymphocytes will be determined by the expression of cell surface differentiation antigens of blood lymphocytes (Simultest IMK-Lymphocyte, Becton Dickinson, San Jose, USA) by flow cytometry (Becton Dickinson, San Jose, USA). 

g) Cytokines (EDTA-plasma). Quantitative analysis of up to 90 different cytokines and acute phase proteins (by FACS bead array technique from 0.1 ml plasma, SCK-CEN Mol/Belgium) will allow to monitor pro- and anti-inflammatory changes in detail and provide useful insight into Th1/Th2 polarization of the immune system. The information on cytokine profiles will further confirm and extent the knowledge from functional assay significantly.
h) Quantitative determination of peroxides in EDTA-plasma as a marker of oxidative metabolic stress (0.1 ml plasma, PerOx-Assay. Immundiagnostik, Germany). Data will be correlated to marker of oxidative stress from breathing air analyses and to oxidative burst.
i) In vitro Delayed Type Hypersensitivity (DTH) Reaction: To monitor cellular immune-reactivity, the in vitro Delayed Type Hypersensitivity (DTH)-reaction is performed by a standardized recall antigen test which contains a mixture of different antigens of viral, bacterial, fungal origin. After addition of whole blood to antigen in vitro, cytokine profiles are measured in the supernatant which will further substantiate results on Th1/Th2 polarization (Immumed, Munich, Germany).
l) mRNA expression: Gene-expression analyses in blood leukocytes are usually limited by small amount of RNA. To overcome this hurdle we use a T7 RNA Polymerase driven transcription of cDNA obtained from total RNA samples to amplify sufficient amounts of aRNA. This amplification method is commonly referred to as the “Eberwine method” (TargetAmp 1-Round aRNA Amplification Kit, Epicentre, Madison, U.S.A.).
Using a real-time-PCR-based array, we will analyze white blood cells also for changes in expression of a focused panel of genes related to inflammation. For this purpose, we will use the Human Inflammatory Cytokines & Receptors RT² Profiler™ PCR Array (Superarray, Frederick, U.S.A.), which profiles the expression of 84 key genes involved in the inflammatory response (Chemokines, cytokines, and interleukins involved in the inflammatory response are represented as well as their receptors).
mRNA expression of cell site receptors that are likely involved in the adenosine (and catecholamine) dependent immune modulation will be analysed by real-time PCR methods using gene specific primers and fluorogenic hydrolysis probes (PrimerDesign Ltd, Southampton, UK).

Evaluation of stress-associated neuro-endocrine regulation:

a) Spielberger's State-Trait Anxiety Inventory is one of the most frequently used questionnaires in trials to investigate the effects of stress-associated psychic effects. This test will be performed prior to and twice after end of the BR period.
b) Current stress test (CST-paper or computerized test): The CST was developed to determine an individuals’ psychological state under the conditions of acute and chronic stress and was validated by German psychologists (Beltz Testzentrale European Test Publisher Group, Göttingen, Germany). The test will be performed at the same time points as saliva collection.
c) Catecholamine: Determination of dopamine, norepinephrine and epinephrine in urine collected within 24 hours (high performance liquid chromatography [HPLC], Chromosytems, Germany) to monitor activation of stress responses, and modulation of immune cell functions through catecholamines [K.J. Tracey, Nature 2002]. 
e) Cortisol (saliva-samples): Saliva is collected in the morning (8 a.m.) and in the evening (8 p.m.) by chewing on a cotton swab for 30 - 45 seconds (SALIVETTE®, Sarstedt, Nürnbrecht, Germany). Absolute values and changes in the circadian rhythm of cortisol secretion (ratio between the morning and evening cortisol values is calculated  = m/e-ratio) are one of the most important indicators of a coordinated stress response. Corticoids are known to modulate stress-associated immune responses and are controlled though adrenocorticotropin (ACTH, ACTH-ELISA, MD Bioscience, Switzerland).
d) Anandamide (endogenous cannabinoid): The endogenous cannabinoids are part of an evolutionary old stress effector system reducing anxiety and enabling individuals to actively remove stressful and painful episodes from their memory. The anandamides will be determined from whole blood (0.1 ml EDTA blood). Blood storage is possible without addition of stabilizing solutions.

Energy metabolism and tissue perfusion:

a) Plasma concentrations of purines: Plasma concentrations of purine nucleosides (adenosine, inosine, hypoxanthine and xanthine) are analysed by HPLC. To abrogate the formation of additional purine metabolites by the degradation of adenine nucleotides blood samples will be withdrawn into syringes pre-filled with inhibitors of ecto-nucleotidases and non-specific phosphatases (“Adenosine Preservation Solution” APS, 1 ml whole blood mixed with 1 ml APS).
b) Lactate: This indicator of an anaerobic metabolism will be determined from EDTA plasma which will be kept frozen until measurement (0.1 ml EDTA plasma).

Immunological and metabolic monitoring by exhaled breath analyses (ongoing):

Based on the experience with clinical studies and results obtained in studies during parabolic flight, we intend for the first time to include molecular breath gas analyses by IMR mass-spectrometry in order to determine biologic markers and bioproducts in exhaled air. The molecular analysis of exhaled breath offers a novel and noninvasive method to monitor oxidative stress and metabolic changes. Offline measurements of exhaled gas are performed simply by collecting expiratory air in vials which can be stored for prolonged time periods. Alternatively, the gas analyzer could become part of the on-site equipment and be used for online measurements (by breathing into a face mask connected to the analyzer).

Category 2 data:

The priority is marked by I (high) and II (low).

a) Resting ECG (I). Why: When the heart rate is recorded during blood pressure measurement (under the rules of Category 3 Data) the priority to get resting ECG is (II). Why: Heart rate and blood pressure are good estimates to monitor the consequences of sympathoadrenal activation.
b) Body weight (I). Why: Parameter can be correlated to urine excretion (total volume, creatinine) and intensity of catabolic cell metabolism resulting in weigth loss.
c) Temperature (II). Why: Supplemental parameters of inflammation and circadian rhythmic regulation.
d) Nitrogen Balance (I). Why: Marker of short-term catabolic, high energy turnover reactions due to STBR and CM (AG).
e) Clinical blood test (I). Why: Baseline data of blood parameters which are crucial for the immune monitoring proposed here.
f) Vitamine, mineral and iron status (I). Why: Important markers related to the degree of oxidative stress (antioxidative role of vitamins, effects of nutritional CMs) and consequences of nutrition and nutritional changes.
g) Additional blood and urine parameters of interest (I). Why: Many urine markers are to be correlated with blood and saliva (e.g. cortisol).
h) Log of Critical incidents (I), PNAS (I), Sleep Assessment (I), Device specific Questions (II) and POMS (II), BDI (II). Why: The documentation of these events is important for the correct interpretation of the data collected during BR.

M. Feuerecker, B. Feuerecker, S. Matzel, M. Long, C. Strewe, I. Kaufmann, M. Hoerl, G. Schelling, M. Rehm, A. Choukèr, (2013), "Five days head down tilt bed rest induces non-inflammatory shedding of L-selectin", Journal of Applied Physiology, 115, 2, pp. 235-242.
A. Choukèr, B. Feuerecker, M. Feuerecker, S. Matzel, I. Kaufmann, C. Strewe, M. Hoerl, G. Schelling, (2013), "Psychoneuroendocrine alterations during 5 days head-down tilt bed rest and artificial gravity interventions", European Journal of Applied Physiology, 113, 8, doi: 10.1007/s00421-013-2640-9, pp. 2057-2065.
M. Feuerecker, B. Feuerecker, S. Matzel, C. Strewe, M. Hoerl, I. Kaufmann, G. Schelling, M. Rehm, A. Choukèr, (2012), "Immune responses during short term bedrest: "Sterile" L-selectin shedding as a marker for acute volume shifts?", Life in Space for Life on Earth Symposium, Aberdeen, United Kingdom, 18-22 June 2012.
B. Feuerecker, M. Feuerecker, S. Matzel, C. Strewe, M. Hoerl, I. Kaufmann, G. Schelling, A. Choukèr, "Testing centrifugation protocols during short term bed rest at 6° HDT: differential psycho-neuro-endocrine responses", Life in Space for Life on Earth Symposium, Aberdeen, United Kingdom, 18-22 June 2012.
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Figure 1: Short- (STBR) [mid- (MTBR) and long-term bedrest (LTBR)] will induce psycho-neuro-endocrine, metabolic and physical stress responses which will all affect the human immune system with increasing intensity.

Figure 2: Countermeasures, as applied by nutrition or physical means (vibration with or without exercise, artificial gravity) will modulate BR induced alterations of the specific (adaptive) and non-specific (innate) parts of the immune system.
© 2020 European Space Agency